CRISPR-Cas9 is revolutionizing the field of genome editing

The transformative CRISPR-Cas9 technology is revolutionizing the field of genome editing. Able to achieve highly flexible and specific targeting, the CRISPR-Cas9 system can be modified and redirected to become a powerful tool for genome editing in broad applications such as stem cell engineering, gene therapy, tissue and animal disease models, and engineering disease-resistant transgenic plants. We've put together a collection of resources that we hope will give you the confidence to get started and to continuously improve your research.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR- associated (Cas) system is the latest addition to the genome editing toolbox, offering a simple, rapid, and efficient solution. Derived from components of a simple bacterial immune system, the CRISPR-Cas9 system permits targeted gene cleavage and gene editing in a variety of eukaryotic cells, and because the endonuclease cleavage specificity in CRISPR-Cas9 system is guided by RNA sequences, editing can be directed to virtually any genomic locus by engineering the guide RNA sequence and delivering it along with the Cas endonuclease to your target cell. The CRISPR-Cas9 system has great promise in broad applications such as stem cell engineering, gene therapy, tissue and animal disease models, and engineering disease-resistant transgenic plants.

The CRISPR-Cas9 system is composed of a short noncoding guide RNA (gRNA) that has two molecular components: a target-specific CRISPR RNA (crRNA) and an auxiliary trans-activating crRNA (tracrRNA). The gRNA unit guides the Cas9 protein to a specific genomic locus via base pairing between the crRNA sequence and the target sequence (Figure 1).

Figure 1. The CRISPR gRNA and Cas9 Protein.

In bacteria CRISPR loci are composed of a series of repeats separated by segments of exogenous DNA (of ~30 bp in length), called spacers. The repeat-spacer array is transcribed as a long precursor and processed within repeat sequences to generate small crRNAs that specify the target sequences (also known as protospacers) cleaved by Cas9 protein, the nuclease component of CRISPR system. CRISPR spacers are then used to recognize and silence exogenous genetic elements at the DNA level. Essential for cleavage is a three-nucleotide sequence motif (NGG) immediately downstream on the 3’ end of the target region, known as the protospacer-adjacent motif (PAM). The PAM is present in the target DNA, but not the crRNA that targets it (Figure 2).

Figure 2. The CRISPR-Cas9 System.

Upon binding to the target sequence, the Cas9 protein induces a specific double-strand break. Following DNA cleavage, the break is repaired by cellular repair machinery through non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms. With target specificity defined by a very short RNA-coding region, the CRISPR-Cas9 system greatly simplifies genome editing (Figure 3).

Figure 3. A CRISPR-Cas9 targeted double-strand break. Cleavage occurs on both strands, 3 bp upstream of the NGG proto-spacer adjacent motif (PAM) sequence on the 3’ end of the target sequence.

Available GeneArt CRISPR-Cas9 genome-editing tools

CRISPR-Cas9 system greatly simplifies genome editing and has great promise in broad applications such as stem cell engineering, gene therapy, tissue and animal disease models, and engineering disease-resistant transgenic plants.

Currently we are the only company to offer the complete suite of genome editing reagents. These gene-editing solutions are paired with optimal cell culture reagents, delivery methods, and analysis tools, based on your application and cell type.

At Thermo Fisher Scientific, we are committed to helping you stay ahead and advance your science through education. Our experienced team has designed a comprehensive four-day CRISPR workshop comprised of both lectures and hands-on laboratory work at our state-of-the-art training facility.

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